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Suspension Culture of Human Embryonic Stem Cells

a technology of stem cells and suspension culture, which is applied in the field of suspension culture of human embryonic stem cells to achieve the effect of rapid and volume-efficien

Active Publication Date: 2010-06-10
ASTERIAS BIOTHERAPEUTICS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0008]This disclosure provides an improved system for culturing and proliferating primate pluripotent stem (hES) cells. The suspension culture system of this invention enables the user to produce high-quality embryonic stem cells in a rapid and volume-efficient mode, for use in therapy and drug discovery.

Problems solved by technology

A significant challenge to the use of pluripotent stem cells for therapy is that they are traditionally cultured on a layer of feeder cells to prevent differentiation (U.S. Pat. No. 5,843,780; U.S. Pat. No. 6,090,622).

Method used

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  • Suspension Culture of Human Embryonic Stem Cells
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  • Suspension Culture of Human Embryonic Stem Cells

Examples

Experimental program
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Effect test

example 1

Growing Pluripotent Stem Cells in Rapid Expansion Medium

[0089]A line of hES cells was obtained that had originally been grown on mouse embryonic fibroblast feeder cells, and then expanded for 20 passages in a feeder-free environment comprising Matrigel® extracellular matrix and conditioned medium, according to U.S. Pat. No. 6,800,480; as elaborated in Xu et al., Stem Cells 2005; 23(3):315-23.

[0090]The hES cells were next weaned onto X-VIVO™ 10 expansion medium from Biowhittaker; or QBSF™-60 from Quality Biological Inc. For use in these experiments, the X-VIVO™ 10 medium was supplemented with the usual goodies: namely, 2 mM L-glutamine, 1% non-essential amino acids (Gibco), 0.1 mM β-mercaptoethanol, and 8 ng / mL bFGF. The medium was further supplemented with 8 ng / mL or 40 ng / mL of bFGF (Gibco); 40 ng / mL of bFGF and 15 ng / mL of SCF (R & D System); or 40 ng / mL of bFGF and 75 ng / mL of Flt3 ligand (R & D System). QBSF™-60 medium was supplemented with 0.1 mM β-mercaptoethanol, 1% non-essen...

example 2

Culture of hES Cells in a Defined System Free of Animal-Based Products

[0098]hES cells cultured in MEF-CM on Matrigel® were passaged to a fresh (non-conditioned) serum free medium: X-VIVO™ 10 supplemented with Glutamine, non-essential amino acids and β-mercaptoethanol, plus 80 ng / mL human basic FGF on Matrigel®, and then adapted to surfaces coated with human laminin. Alternatively, cryopreserved cells were directly thawed into the same medium containing 80 ng / mL hbFGF. The cells were passaged every 5-6 days using Collagenase IV.

[0099]Cultures grown under these conditions were similar or better than cultures on Matrigel®. (A) morphology for cells grown in mEF conditioned medium; (B) morphology fin defined medium on laminin; (C) Surface marker SSEA-4 expression in mEF-CM (H1p62) or defined medium (H1p34+28); (D) Expression of surface marker Tra-1-60 in mEF-CM or defined medium. Culture performance in the defined medium on laminin was superior: very large ES cell colonies were observed,...

example 3

Culturing hES Cells in Suspension

[0102]With a view to increasing the yield of hES cells per culture volume, the cells were cultured in suspension, and then evaluated for morphology and their ability to form differentiated cells representative of all three germ layers.

[0103]H9 hES cells grown on Matrigel® were harvested from 6-well plates and seeded into a spinner flask under the following condition[0104]Vessel: 100 mL spinner flask[0105]Inoculation (seeding) density: 3.6×105 cells / mL;[0106]Medium volume: 50 mL per spinner flask[0107]Medium used: mEF conditioned medium containing bFGF (8 ng / mL)[0108]Agitation rate: 20 rpm (Bellco carrier magnetic stirrer)[0109]Atmosphere: 37° C. CO2 incubator[0110]Medium exchange: Every other day (exchanged by letting the cells settle down and replacing the supernatant)

H9 hES cells were maintained in the spinner flask under these conditions for 6 days.

[0111]FIG. 4 (upper panel) shows the results. Following an initial decline during which the culture ...

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Abstract

This disclosure provides an improved system for culturing human embryonic stem cells. The cells are cultured in suspension so as to maximize the production capacity of the culture environment. The new culture system of this invention allows for bulk proliferation of hES cells in a more cost-effective manner, which facilitates commercial production of important products for use in human therapy.

Description

OTHER PATENT APPLICATIONS[0001]This application claims priority to U.S. Ser. No. 60 / 693,266, filed Jun. 22, 2005.BACKGROUND[0002]Regenerative medicine is benefiting from recent advances relating to the isolation, culture, and use of various types of progenitor cells. This disclosure provides further improvements for the commercial development of human pluripotent stem cells and their derivatives.[0003]Embryonic stem cells have two very special properties: First, unlike other normal mammalian cell types, they can be propagated in culture almost indefinitely, providing a virtually unlimited supply. Second, they can be used to generate a variety of tissue types of interest as a source of replacement cells and tissues for use in tissue therapy, or for use in the screening of pharmaceutical agents.[0004]Thomson et al. (U.S. Pat. No. 5,843,780; Proc. Natl. Acad. Sci. USA 92:7844, 1995) were the first to successfully isolate and propagate pluripotent stem cells from primates. They subseque...

Claims

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Application Information

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IPC IPC(8): C12N5/0735
CPCC12N5/0606C12N2501/115C12N2501/26C12N2501/15C12N2501/125C12N2533/52C12N2501/119C12N2501/113
Inventor MANDALAM, RAMKUMARLI, YANNADEAU-DEMERS, ISABELLE
Owner ASTERIAS BIOTHERAPEUTICS INC
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